TRAF family proteins interact with the common neurotrophin receptor and modulate apoptosis induction.

The common neurotrophin receptor, p75(NTR), has been shown to signal in the absence of Trk tyrosine kinase receptors, including induction of neural apoptosis and activation of NF-kappaB. However, the mechanisms by which p75(NTR) initiates these intracellular signal transduction pathways are unknown. Here we report interactions between p75(NTR) and the six members of TRAF (tumor necrosis factor receptor-associated factors) family proteins. The binding of different TRAF proteins to p75(NTR) was mapped to distinct regions in p75(NTR). Furthermore, TRAF4 interacted with dimeric p75(NTR), whereas TRAF2 interacted preferentially with monomeric p75(NTR). TRAF2-p75(NTR), TRAF4-p75(NTR), and TRAF6-p75(NTR) interactions modulated p75(NTR)-induced cell death and NF-kappaB activation with contrasting effects. Coexpression of TRAF2 with p75(NTR) enhanced cell death, whereas coexpression of TRAF6 was cytoprotective. Furthermore, overexpression of TRAF4 abrogated the ability of dimerization to prevent the induction of apoptosis normally mediated by monomeric p75(NTR). TRAF4 also inhibited the NF-kappaB response, whereas TRAF2 and TRAF6 enhanced p75(NTR)-induced NF-kappaB activation. These results demonstrate that TRAF family proteins interact with p75(NTR) and differentially modulate its NF-kappaB activation and cell death induction.

Lymphotoxin-beta receptor signaling complex: role of tumor necrosis factor receptor-associated factor 3 recruitment in cell death and activation of nuclear factor kappaB.

The binding of heterotrimeric lymphotoxin, LT alpha1 beta2, to the LTbeta receptor (LTbeta R), a member of the tumor necrosis factor receptor (TNFR) superfamily, induces nuclear factor kappaB (NF-kappaB) activation and cell death in HT29 adenocarcinoma cells. We now show that treatment with LT alpha1 beta2 or agonistic LTbeta R antibodies causes rapid recruitment of TNFR-associated factor 3 (TRAF3) to the LTbeta R cytoplasmic domain. Further, stable overexpression of a TRAF3 mutant that lacks the RING and zinc finger domains inhibits LTbeta R-mediated cell death. The inhibition is specific for LTbeta R cell death signaling, since NF-kappaB activation by LT alpha1 beta2 and Fas-mediated apoptosis are not inhibited in the same cells. The mutant and endogenous TRAF3s are both recruited at equimolar amounts to the LTbeta R, suggesting that the mutant disrupts the function of the signaling complex. These results implicate TRAF3 as a critical component of the LTbeta R death signaling complex and indicate that at least two independent signaling pathways are initiated by LTbeta R ligation.

Hepatitis C virus core protein interacts with the cytoplasmic tail of lymphotoxin-beta receptor.

Hepatitis C virus (HCV) core protein is a multifunctional protein. We examined whether it can interact with cellular proteins, thus contributing to viral pathogenesis. Using the HCV core protein as a bait to screen a human liver cDNA library in a yeast two-hybrid screening system, we have isolated several positive clones encoding cellular proteins that interact with the HCV core protein. Interestingly, more than half of these clones encode the cytoplasmic domain of lymphotoxin-beta receptor (LT betaR), which is a member of the tumor necrosis factor receptor family. Their binding was confirmed by in vitro glutathione S-transferase fusion protein binding assay and protein-protein blotting assay to be direct and specific. The binding sites were mapped within a 58-amino-acid region of the cytoplasmic tail of LT betaR. The binding site in the HCV core protein was localized within amino acid residues 36 to 91 from the N terminus, corresponding to the hydrophilic region of the protein. In mammalian cells, the core protein was found to be associated with the membrane-bound LT betaR. Since the LT betaR is involved in germinal center formation and developmental regulation of peripheral lymphoid organs, lymph node development, and apoptotic signaling, the binding of HCV core protein to LT betaR suggests the possibility that this viral protein has an immunomodulating function and may explain the mechanism of viral persistence and pathogenesis of HCV.

Immunohistochemical analysis of in vivo patterns of TRAF-3 expression, a member of the TNF receptor-associated factor family.

An immunohistochemical approach was used to explore the in vivo expression of TNF receptor-associated factor 3 (TRAF-3), a putative signaling protein that binds to the cytosolic domains of CD30, CD40, and lymphotoxin-beta receptors. TRAF-3 immunostaining was detected in many types of cells throughout the human body. TRAF-3 immunostaining was only rarely present in thymocytes but was found in the thymic epithelioreticular cells. Lymphocytes in the bone marrow were also typically TRAF-3 immunonegative, whereas myeloid progenitor cells and megakaryocytes were often TRAF-3 positive. Peripheral blood lymphocytes were mostly TRAF-3 immunonegative, while granulocytes were TRAF-3 immunopositive. Monocytes were strongly immunostained for TRAF-3, but macrophages in nodes typically contained little or no TRAF-3 immunoreactivity. Some lymphocytes within the germinal centers of secondary lymphoid follicles in normal and reactive nodes were TRAF-3 immunopositive, as were occasional interfollicular lymphocytes in the T cell regions of these organs, but most lymphocytes appeared to be TRAF-3 immunonegative or stained only weakly. Plasma cells, however, were strongly TRAF-3 positive. Stimulation of PBLs with anti-CD3 Ab induced marked increases in the steady state levels of TRAF-3 protein in vitro as determined by immunoblotting, while levels of TRAF-2 were unchanged, implying a dynamic regulation of TRAF-3 expression. The findings establish for the first time the cell type- and differentiation-specific patterns of expression of a member of the TRAF family of proteins.

Apoptosis mediated by the TNF-related cytokine and receptor families.

T lymphocytes use several specialized mechanisms to induce apoptotic cell death. The tumor necrosis factor (TNF)-related family of membrane-anchored and secreted ligands represent a major mechanism regulating cell death and cell survival. These ligands also coordinate differentiation of tissue to defend against intracellular pathogens and regulate development of lymphoid tissue. Cellular responses are initiated by a corresponding family of specific receptors that includes two distinct TNFR (TNFR60 and TNFR80), Fas (CD95), CD40, p75NTF, and the recently identified lymphotoxin beta-receptor (LT beta R), among others. The MHC-encoded cytokines, TNF and LT alpha, form homomeric trimers, whereas LT beta assembles into heterotrimers with LT alpha, creating multimeric ligands with distinct receptor specificities. The signal transduction cascade is initiated by transmembrane aggregation (clustering) of receptor cytoplasmic domains induced by binding to their multivalent ligands. The TRAF family of Zn RING/finger proteins bind to TNFR80; CD40 and LT beta R are involved in induction NF kappa B and cell survival. TNFR60 and Fas interact with several distinct cytosolic proteins sharing the "death domain" homology region. TNF binding to TNFR60 activates a serine protein kinase activity and phosphoproteins are recruited to the receptor forming a multicomponent signaling complex. Thus, TNFRs use diverse sets of signaling molecules to initiate and regulate cell death and survival pathways.

The Epstein-Barr virus transforming protein LMP1 engages signaling proteins for the tumor necrosis factor receptor family.

The cytoplasmic C-terminus of Epstein-Barr virus (EBV) latent infection membrane protein 1 (LMP1) is essential for B lymphocyte growth transformation and is now shown to interact with a novel human protein (LMP1-associated protein 1 [LAP1]). LAP1 is homologous to a murine protein, tumor necrosis factor receptor-associated factor 2 (TRAF2), implicated in growth signaling from the p80 TNFR. A second novel protein (EBI6), induced by EBV infection, is the human homolog of a second murine TNFR-associated protein (TRAF1). LMP1 expression causes LAP1 and EBI6 to localize to LMP1 clusters in lymphoblast plasma membranes, and LMP1 coimmunoprecipitates with these proteins. LAP1 binds to the p80 TNFR, CD40, and the lymphotoxin-beta receptor, while EBI6 associates with the p80 TNFR. The interaction of LMP1 with these TNFR family-associated proteins is further evidence for their role in signaling and links LMP1-mediated transformation to signal transduction from the TNFR family.

TNF receptor signal transduction. Ligand-dependent stimulation of a serine protein kinase activity associated with (CD120a) TNFR60.

TNF is a pluripotent cytokine that mediates activities through two distinct receptors of 55 to 60 kDa (CD120a, known as TNFR60) and 75 to 80 kDa (CD120b, known as TNFR80). These receptors share homology in the extracellular ligand binding region; however, the cytoplasmic domains are distinct and lack any inherent enzymatic activity, which suggests that ligand binding and subsequent receptor clustering leads to the association of active signaling molecules with TNFRs. To test this hypothesis, we isolated TNFRs by immunoprecipitation and examined the immune complexes for the presence of associated phosphoproteins and protein kinase activity. In the U-937 monocytic cell line, prelabeled with 32PO4, TNF induces the association of several phosphoproteins with TNFR60, but not TNFR80. The TNFR60 immune complexes also contain a TNF-dependent serine protein kinase activity, which was detected by an in vitro kinase assay, that phosphorylates proteins of 125, 97, 85, and 60 kDa, which are of apparent molecular masses that are similar to those of TNF-induced phosphoproteins that coprecipitate with TNFR60. Association of serine protein kinase activity with TNFR60 is rapid and dependent on the concentration of TNF. Proteins of molecular mass similar to the 125- and 97-kDa protein kinase substrates seem to be associated with TNFR60 immune complexes only after exposure of U-937 cells to TNF. The TNFR60-associated protein kinase activity is inhibited by staurosporine, but not by the protein kinase A and C inhibitors, HA-1004 and H7. Staurosporine greatly enhanced the sensitivity of U-937 cells to the cytotoxic effect of TNF. These results suggest a serine protein kinase(s), and, possibly, other TNF-dependent TNFR60-associated proteins may be involved in mediating signals through TNFR60 in response to ligand binding.

A lymphotoxin-beta-specific receptor.

Tumor necrosis factor (TNF) and lymphotoxin-alpha (LT-alpha) are members of a family of secreted and cell surface cytokines that participate in the regulation of immune and inflammatory responses. The cell surface form of LT-alpha is assembled during biosynthesis as a heteromeric complex with lymphotoxin-beta (LT-beta), a type II transmembrane protein that is another member of the TNF ligand family. Secreted LT-alpha is a homotrimer that binds to distinct TNF receptors of 60 and 80 kilodaltons; however, these receptors do not recognize the major cell surface LT-alpha-LT-beta complex. A receptor specific for human LT-beta was identified, which suggests that cell surface LT may have functions that are distinct from those of secreted LT-alpha.

Production of lymphotoxin (LT alpha) and a soluble dimeric form of its receptor using the baculovirus expression system.

Human LT alpha and a fusion protein (p60:Fc) comprised of the extracellular domain of the 60 kDa TNF receptor (TNFR60) fused to the Fc portion of human IgG1 were produced in insect cells infected with recombinant baculoviruses. The p60:Fc fusion produced in insect cells accumulates in culture supernatants to levels > 2 mg/l. Purified p60:Fc binds human TNF and LT alpha with high affinity (200-600 pM) and neutralizes TNF cytolytic activity at equimolar stoichiometric concentration. The data show that p60:Fc is an effective ligand-precipitating reagent which recognizes recombinant LT alpha produced in mammalian or insect cells and naturally occurring LT alpha produced in T cells. The levels of human LT alpha produced in baculovirus-infected insect cells is estimated to be approximately 20 mg/l. Insect cell-derived human LT alpha is biologically active in an L929 cytotoxicity assay and is efficiently neutralized by p60:Fc. These data demonstrate that the baculovirus system is useful for overexpressing biologically active LT alpha and p60:Fc and therefore, may be applicable to other oligomeric cytokines and soluble dimeric cytokine receptors.

Specific induction of 80-kDa tumor necrosis factor receptor shedding in T lymphocytes involves the cytoplasmic domain and phosphorylation.

The 80-kDa TNFR (TNFR80) expressed by activated human T cells is constitutively phosphorylated and undergoes limited proteolytic cleavage (shedding) at the cell surface releasing a 40-kDa soluble TNF-binding protein. Triggering of activated T cells through the TCR rapidly increased the rate of TNFR80 shedding > 20-fold more than nonstimulated cells, demonstrating that shedding is a specific, inducible process. The protein kinase inhibitor staurosporine inhibited constitutive phosphorylation and blocked inducible shedding of TNFR80, suggesting that phosphorylation may be important for cleavage of the extracellular domain. However, a deletion mutation of the entire cytoplasmic domain of human TNFR80 was shed when expressed in murine L929 cells, albeit relatively poorly compared with full length receptor. This demonstrates that the cytoplasmic domain is important but not essential for cleavage of the extracellular domain of TNFR80. Moreover, a requirement for phosphorylation of proteins other than the receptor was revealed by the finding that staurosporine completely blocked cleavage of the cytoplasmic deletion mutant. Collectively, these results demonstrate that protein phosphorylation is essential and the cytoplasmic domain is important for regulating the inducible production of soluble TNF-binding proteins by activated effector T cells.

Tumor necrosis factor (TNF) receptor expression in T lymphocytes. Differential regulation of the type I TNF receptor during activation of resting and effector T cells.

The expression of TNF-alpha receptors (TNFR) was examined on a CD4+ T cell hybridoma, transformed T cell lines, CTL clones, and activated T cells from peripheral blood to determine the basis of the immunomodulatory activity of TNF on T cell function. Analyses by ligand cross-linking and competitive binding assays with mAb to the 80-kDa receptor (TNFR-I), demonstrated that the TNFR-I was the predominant receptor expressed on activated CD4+ and CD8+ T cell subsets. However, on T cell leukemic lines, a second, non-TNFR-I binding site was identified, most likely the 55-kDa form (TNFR-II). Additional subsets of T cells were readily distinguished by their expression of TNFR-I and related members of the TNFR gene family (CD40 and CD27). Expression of the TNFR-I was dependent upon the state of T cell activation. Signaling through the TCR for Ag or IL-2R was sufficient to induce TNFR mRNA and protein expression in resting T cells. Multiple sizes of TNFR-I transcripts were detected during T cell activation; however, biosynthetic studies showed these multiple species encode a single protein of 80 kDa. These results, combined with the known ability of TNF to induce IL-2R expression, indicate that TNF and IL-2 form a reciprocating receptor amplification circuit. In contrast, differentiated effector T cells triggered through the TCR or protein kinase C initiated a rapid down-regulation (transmodulation) of the TNFR-I that preceded TNF or lymphotoxin secretion. The mechanism of transmodulation involved proteolytic processing of the mature 80-kDa receptor releasing a soluble 40-kDa fragment. This indicates that a TNF autocrine loop is not likely to form during the response of an effector T cell. Collectively, these results suggest that transcriptional and post-translational modification of the TNFR-I are important control points regulating the expression of this receptor during T cell activation.